Targeting Extracellular Domains D4 and D7 of Vascular Endothelial Growth Factor Receptor 2 Reveals Allosteric Receptor Regulatory Sites
- 1 October 2012
- journal article
- review article
- Published by Taylor & Francis Ltd in Molecular and Cellular Biology
- Vol. 32 (19), 3802-3813
- https://doi.org/10.1128/mcb.06787-11
Abstract
Vascular endothelial growth factors (VEGFs) activate three receptor tyrosine kinases, VEGFR-1, -2, and -3, which regulate angiogenic and lymphangiogenic signaling. VEGFR-2 is the most prominent receptor in angiogenic signaling by VEGF ligands. The extracellular part of VEGF receptors consists of seven immunoglobulin homology domains (Ig domains). Earlier studies showed that domains 2 and 3 (D23) mediate ligand binding, while structural analysis of dimeric ligand/receptor complexes by electron microscopy and small-angle solution scattering revealed additional homotypic contacts in membrane-proximal Ig domains D4 and D7. Here we show that D4 and D7 are indispensable for receptor signaling. To confirm the essential role of these domains in signaling, we isolated VEGFR-2-inhibitory "designed ankyrin repeat proteins" (DARPins) that interact with D23, D4, or D7. DARPins that interact with D23 inhibited ligand binding, receptor dimerization, and receptor kinase activation, while DARPins specific for D4 or D7 did not prevent ligand binding or receptor dimerization but effectively blocked receptor signaling and functional output. These data show that D4 and D7 allosterically regulate VEGFR-2 activity. We propose that these extracellular-domain-specific DARPins represent a novel generation of receptor-inhibitory drugs for in vivo applications such as targeting of VEGFRs in medical diagnostics and for treating vascular pathologies.Keywords
This publication has 30 references indexed in Scilit:
- Signal transduction by vascular endothelial growth factor receptorsBiochemical Journal, 2011
- An Antibody Targeted to VEGFR-2 Ig Domains 4-7 Inhibits VEGFR-2 Activation and VEGFR-2–Dependent Angiogenesis without Affecting Ligand BindingMolecular Cancer Therapeutics, 2011
- Structural determinants of growth factor binding and specificity by VEGF receptor 2Proceedings of the National Academy of Sciences of the United States of America, 2010
- Direct contacts between extracellular membrane-proximal domains are required for VEGF receptor activation and cell signalingProceedings of the National Academy of Sciences of the United States of America, 2010
- Transmembrane domain‐mediated orientation of receptor monomers in active VEGFR‐2 dimersThe FASEB Journal, 2009
- Structure and function of VEGF receptorsIUBMB Life, 2009
- Contacts between membrane proximal regions of the PDGF receptor ectodomain are required for receptor activation but not for receptor dimerizationProceedings of the National Academy of Sciences of the United States of America, 2008
- In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitroNature Protocols, 2007
- Insights into ErbB signaling from the structure of the ErbB2-pertuzumab complexCancer Cell, 2004
- Characteristics of a Human Cell Line Transformed by DNA from Human Adenovirus Type 5Journal of General Virology, 1977